JP2021519800A - Method for producing membrane protein - Google Patents

Abstract

A method for producing a membrane protein including the following steps is disclosed: expressing the membrane protein in a host organism present in an aqueous medium, releasing the membrane protein from the host organism, adding a surfactant solution. To solubilize the membrane protein, recover the liquid fraction of the solubilized membrane protein, subject the liquid fraction to chromatography to bind or retain the membrane protein on a stationary phase, and use an elution buffer. To produce the membrane protein by eluting the stationary phase. This method can produce a relatively large amount of membrane protein in an efficient manner without compromising the quality of the final product.

Description

The present invention relates to a method for producing a membrane protein. The method according to the invention is suitable for industrial use where large amounts of membrane proteins are produced. Therefore, this method is intended to apply unit operations that are at least suitable for the production of pilot plants. In particular, the present invention aims to avoid ultracentrifuges.

About one-third of the genes in the human genome encode membrane proteins. Membrane proteins affect many important cell activities such as energy conversion, cell signaling, cell-cell interaction, cell adhesion, cell migration, protein transport, viral fusion, neurosynaptic activity, ion and metabolite transport. .. Membrane proteins are embedded in the lipid bilayer of cell membranes and are composed of both hydrophobic and hydrophilic sites.

In recent years, membrane proteins have succeeded in fusing with a thin film composite layer transplanted on a porous structure support layer. In addition, membrane proteins represent important pharmaceutical targets and interesting research subjects in cell biology and protein biochemistry. Therefore, there is a need for large-scale production of pilot plants or membrane proteins.

WO2017137361 (A1) discloses self-assembled nanostructures formed between transmembrane proteins such as aquaporin water channels (AQPs) and polyalkyleneimines (PAIs). The self-assembled nanostructures are then incorporated into a thin film composite (TFC) membrane implanted in a porous support membrane. The porous support membrane can be a hollow fiber, a flat sheet, a spiral wrap membrane, etc. for reverse osmosis or forward osmosis.

WO 2013/043118 discloses a thin film composite (TFC) membrane in which aquaporin water channels (AQP) are incorporated into the active layer of the membrane. In addition, it discloses methods for producing thin film composites and their use in filtration methods such as nanofiltration and osmotic filtration methods. The TFC membrane is composed of lipid-AQP / copolymer-AQP vesicles incorporated into the TFC active layer. WO2010 / 146365 describes the preparation of TFC-aquaporin-Z (AqpZ) filtration membranes using amphipathic triblock copolymers as vesicle-forming materials for incorporating immobilized AQP.

WO2014 / 108827 discloses a hollow fiber (HF) module having fibers modified with a thin film complex (TFC) layer containing aquaporin water channels that are incorporated into vesicles before the aquaporin water channels are incorporated into the TFC layer.

Industrial methods for isolating large amounts of membrane proteins from cells are usually not available to those of skill in the art. Previous techniques have focused on the production of relatively small amounts of membrane proteins for research purposes. Therefore, relatively cumbersome and labor-intensive manufacturing methods have been tolerated. However, recent higher demands for membrane proteins in industrial membranes for reverse osmosis and forward osmosis have revealed the need for more efficient production methods. An object of the present invention is to provide a method for producing a membrane protein, which can produce a relatively large amount of membrane protein in an efficient manner without compromising the quality of the final product.

The present invention relates to a method for producing a membrane protein, and the production method is described in the following steps:
a. Expressing membrane proteins in a host organism present in an aqueous medium,
b. To release the membrane protein from the host organism,
c. To solubilize the membrane protein by adding a detergent solution,
d. Collecting the liquid fraction of the solubilized membrane protein as a supernatant by centrifugation,
e. The chromatographic liquid fraction is subjected to chromatography to bind or retain the membrane protein on a stationary phase, and
f. To produce the membrane protein by eluting the stationary phase with an elution buffer.
Here, the centrifugation of step d is carried out at 500 g to 30,000 g.

The method of the present invention has the advantage of being able to handle large amounts of aqueous media containing said host organisms. Therefore, the method can process 50 L or more quantities, such as 100 L or more, by applying unit operations suitable for pilot plants or full-scale production. In addition, this method is scale-enhancing and can be easily adapted to large amounts of aqueous media containing said host organisms.

Surprisingly, we have found that the addition of detergent solutions solubilizes membrane proteins expressed by host organisms to a higher degree than other proteins. It is estimated that at least about 60% of the proteins in the vesicles formed by the detergent solution are the membrane proteins of interest. In addition, the vesicles formed with the surfactant have been observed to be larger than normal, eg, about 0.5 μm, and the membrane protein is more surfactant than the naturally used surfactant lipids. It suggests that it forms a more stable superform.

The aqueous medium containing the host organism can be used directly in the method. However, in order to obtain a purer product and avoid handling excess amounts of the aqueous medium, the aqueous medium containing the host organism of step a is usually filtered according to step b prior to release of the membrane protein. ..

Generally, the aqueous medium containing the host organism is concentrated by filtering through a filter with pores small enough for cell debris and medium to pass through while the cells are retained. In a suitable embodiment, the aqueous medium containing the host organism of step a is filtered through a microfiltration membrane having a pore size of 0.5 micrometers or less. Preferably, the pore diameter is 0.1 μm or less.

After a filtration step properly performed by microfiltration, the host organism can be isolated from the rest of the aqueous medium. Although several possible separation methods are possible, it is generally preferred to isolate the host organism after filtration by centrifugation of the aqueous medium containing said host organism. The centrifugation method is usually performed with G-force to form pellets within a suitable time. Therefore, the centrifugation is usually carried out in an amount of 500 g or more, for example, 1000 g or more, preferably 2000 g or more. The density of the pellets should not be excessively high to prevent problems in later steps. Therefore, centrifugation is usually not performed in excess of 30,000 g, such as not exceeding 20,000 g, appropriately not exceeding 15,000 g, preferably not exceeding 8000 g.

The cells are collected as pellets and the supernatant is discarded. It is preferred that the isolated host organism be washed with isotonic saline to dissolve the contaminating salt and then centrifuged as described above to isolate the washed host organism. The used cleaning liquid that appears in the supernatant can be discarded.

Pellets containing washed cells can be cryopreserved at −20 ° C. or used directly in the next step. Appropriately, dilution buffer is added prior to step b. The dilution buffer contains a protease inhibitor to prevent degradation of membrane proteins, pH regulators such as TRIS and phosphate to keep pH values within the desired range, and / or ion scavengers such as EDTA. obtain.

The membrane protein can be released from the host organism in several ways, preferably by chemical or mechanical lysis of cells. If the cells are properly chemically lysed, a lysing aqueous solution is added to release the membrane protein from the host organism. In a preferred embodiment of the invention, the aqueous lysis buffer is a detergent solution that simultaneously solubilizes membrane proteins.

Due to cell lysis and membrane protein solubilization, host cells are generally subject to the action of detergents during agitation. In general, the host cell can react with the detergent for at least 1 hour, eg, 2 hours, preferably at least 6 hours.

When mechanical lysis of cells is used, the release of membrane proteins from host cells is generally carried out by a homogenizer. For milk homogenization, it is suitable to use the same type of homogenizer used in the dairy industry. A suitable example is the Stansted 7575 homogenizer. After treatment with a homogenizer, the cells break open and release membrane proteins.

After the release of the membrane protein, a cationic flocculant can be added. Cationic flocculants are believed to interact with, among other things, negatively charged cell debris, thereby forming flocs. In a preferred embodiment of the invention, the cationic flocculant is a polyamine compound such as Superfloc C581.

Flock formation usually does not occur immediately. Therefore, it is preferred that the cationic flocculant can react with the cellular portion of the resuspension to form flocs during gentle agitation. Usually, the cationic flocculant and the cell debris can interact for 10 minutes to 2 hours with stirring at room temperature.

When using chemical lysis of cells, the liquid fraction of step e is recovered as a supernatant from centrifugation of the suspension containing flocs. When the membrane protein is solubilized by the detergent solution, the membrane protein appears in the supernatant.

When mechanical lysis of cells is used, the liquid fraction of step e is recovered from the resuspension of the solid fraction and the solid fraction is resuspended in a detergent solution for solubilizing the membrane protein. In one embodiment of the invention, a solid fraction is formed due to the fact that cell fragments containing membrane proteins interact with flocculants to form flocs, which can be separated from the liquid by centrifugation. NS. In another embodiment of the invention, surprisingly, it has been found that it is possible to collect membrane proteins in pellets when mechanical lysis of cells is used without the use of flocculants. Was done. The pellet obtained by centrifugation can be resuspended in a detergent solution to solubilize the membrane protein. In this step, the liquid fraction of step e is recovered as supernatant by centrifugation.

Centrifugal methods are usually performed with G-force to form pellets in a reasonable amount of time. Therefore, the centrifugation is usually carried out in an amount of 500 g or more, for example, 1000 g or more, preferably 2000 g or more. The density of the pellets should not be excessively high to prevent problems in later steps. Therefore, centrifugation is usually not performed in excess of 30,000 g, such as not exceeding 20,000 g, appropriately not exceeding 10,000 g, preferably not exceeding 8,000 g. When a g-force of less than 30,000 g is applied, a clarifying agent commonly used in dairy products can be used, such as a spore removal centrifuge for GEA, Tetra Pak, Alfa Laval, SPX Flow Seatal Separation Technology. Clarifying agents useful in the present invention may also be referred to by some manufacturers as Bactofuge. Therefore, the present invention may omit the application of ultracentrifuges, which are capable of batch centrifugation and only small amounts of treatment.

Detergents used in the present invention include n-dodecyl-β-D-maltopyranoside (DDM), n-decyl-β-D-maltopyranoside (DM), or 5-cyclohexylpentyl β-D-maltoside (Cymal-5). ) And the like; alkyl glucopyranosides such as n-octyl-β-D-glucopyranoside (OG); amine oxides such as n-lauryldimethylamine N-oxide (LDAO); n-dodecylphosphocholine (FC) -12), phosphocholine such as n-tetradecylphosphocholine (FC-14), or n-hexadecylphosphocholine (FC-16); or polyoxyethylene glycol. In a suitable embodiment of the invention, the surfactant is selected from the group consisting of lauryldimethylamine N-oxide (LDAO), octyl glucoside (OG), dodecyl maltoside (DDM) or a combination thereof. .. LDAO is a preferred surfactant because it produces large and stable vesicles, suggesting that it can replace naturally occurring phospholipids.

The liquid fraction is subjected to a chromatographic method in which the membrane protein is bound or retained in the stationary phase. The type of chromatography can be selected as affinity chromatography, ion exchange chromatography, size exclusion chromatography, substitution chromatography, liquid chromatography, high performance liquid chromatography, reverse phase chromatography, hydrophobic interaction chromatography and the like. Chromatography methods are usually preparative chromatography as opposed to analytical chromatography to form the purification of membrane proteins.

In a currently preferred embodiment, the chromatography method is selected as affinity chromatography, wherein the first portion of the affinity pair is associated with the membrane protein and the second portion of the affinity pair is associated with the stationary phase. Be done. Examples of stationary phases include beads and column materials. In a preferred embodiment of the invention, a stationary phase associated with the second portion of the affinity pair is present on the column. Usually, the portion of the affinity pair is associated with a covalent bond to the membrane protein or the stationary phase. However, it can also be other types of associations such as hybridization, affinity binding via antibody-antigen interactions.

Many affinity pairs applicable to the present invention are known to those skilled in the art and are biotin-streptavidin pairs, antibody-antigen pairs, antibody-haptempene pairs, aptamer affinity pairs, capture protein pairs, Fc receptor-IgG pairs. , Metal chelated lipid pairs, metal chelated lipid-histidine (HIS) tagged protein pairs, or combinations thereof. In a currently preferred embodiment, the affinity pair is a metal chelating lipid-histidine (HIS) tagged protein pair.

The metal is usually immobilized on the column material by a technique called immobilized metal affinity chromatography (IMAC). The metal is usually selected as Cu (II) or Ni (II), preferably Ni (II). Ni-NTA resins can be used to purify His-tagged proteins automatically or manually. Suitable stationary phases are GE Healthcare's "CaptoCharating" or GE Healthcare's HisTrap Gel filtration material (Ni Sepharose 6 Fast Flow).

The first portion of the affinity pair, which is a histidine tag, is usually attached to the C-terminus of the membrane protein. The histidine tag usually contains 6 consecutive histidine amino acids, but in the present invention, it is preferable that the histidine tag contains 8 or more histidine molecules. Due to the large number of histidine amino acids in the histidine tag, specific binding proteins and non-specific binding proteins can be effectively separated.

After adding the liquid fraction containing the membrane protein to the column, the liquid can penetrate the resin by gravity or pressure. Appropriately, the elution buffer comprises imidazole. The imidazole has the ability to interact with the binding of His tags to the metal ions immobilized on the resin. At a specific concentration of imidazole, the His-tagged membrane protein is released.

To separate non-specific binding membrane proteins from the membrane protein of interest, the column prior to elution with elution buffer is generally washed with a wash buffer containing 40% or less of the concentration of imidazole in the elution buffer. .. The concentration of imidazole in the elution buffer is generally in the range of 200 mM to 2000 mM imidazole. In a preferred embodiment of the present invention, the concentration of imidazole in the elution buffer is 400 mM or more. To obtain a more effective elution of the His-tagged membrane protein, the concentration of imidazole in the buffer is typically 600 mM or 800 mM or higher.

For most applications, His tags generally have little effect on protein structure, function, or immunogenicity. However, in certain applications, it may be desirable to remove the His tag after performing the function of binding the membrane molecule to the column. The His tag can be removed by introducing a cleavable link between the membrane protein and the His tag. Suitable cleavable links include pH sensitive linkers, disulfide linkers, protease sensitive linkers, and beta glucuronide linkers.

Membrane proteins extend from the entire bile membrane, the inside of the cell, to the extracellular space in its natural environment. Many transmembrane proteins act as gateways for specific substances, thereby allowing the exchange of these substances between the inside of the cell and the extracellular fluid. A characteristic of transmembrane proteins is the presence of hydrophobic regions, which ensure that transmembrane proteins are integrated into the membrane. The transmembrane protein further has hydrophilic segments on either side of the hollow fiber region, which are directed to the intracellular and extracellular fluids, respectively.

Although it is believed that any membrane protein can be produced according to the present invention, it is generally desirable to use this method to produce a membrane protein that transports ions (ion channels) and water (aquaporin water channels). Ion channels include chloride channels and metal ion transporters. In addition to the chloride ion, the particular chloride channels also _{^{HCO 3 -, I -, SCN}} -, and NO ₃ ^- and conducts. Examples of the metal ion transporter include magnesium transporters, potassium ion channels, sodium ion channels, calcium channels, proton channels and the like. In certain embodiments of the invention, the membrane protein is outer membrane protein A (OmpA).

In a preferred embodiment of the invention, the membrane protein is an aquaporin water channel. Aquaporin water channels facilitate the transport of water into and out of cells. In industrial membranes, the water channels of the aquaporin ensure water flow by permeation, but other components in the solution are excluded. Membrane proteins such as aquaporin water channels can emanate from a variety of sources such as prokaryotes and eukaryotes. As a prokaryotic source of aquaporin, E.I. colli, Kyrpidia spormannii, Methanothermovator sp. , Novivacillus thermophilus, Saccharomyces cerevisiae, and Halomonas sp. Eukaryotic sources of aquaporin include Oryza sativa Japanica (Japanese rice), Eucalyptus grandis, Solanum tuberosum (Denmark potato), and Milnesium tardigradum (tardigrade).

The nuclear sequences of membrane proteins from the source organism are generally codon-optimized using the services of Geneart (a subsidiary of Thermo Fisher Scientific) to improve expression in the host organism. The obtained gene is appropriately synthesized by adding a codon encoding histidine to the C-terminal and adding adjacent restriction sites to the N-terminal and C-terminal. The synthetic gene fragment can be digested with a restriction enzyme and ligated into a vector fragment. The resulting ligation mixture is preferably transformed into an organism such as Escherichia coli DH10B. Antibiotic resistant transformants are appropriately selected on media with antibiotics. Transformants were confirmed by sequencing the gene construct. Subsequently, the isolated vector DNA was transferred to the production host. The production host can be selected from several suitable prokaryotes or eukaryotes such as Escherichia coli and Saccharomyces cerevisiae.

The host organism is generally engineered to express more naturally occurring membrane proteins than normal, or to express non-natural membrane proteins. One method of expressing a membrane protein can be by transforming the host organism with a vector containing the DNA encoding the membrane protein, as described above. Another possibility for obtaining overexpression of membrane proteins would be, for example, by weakening the repressor or upregulating the expression of native membrane proteins by inserting appropriate promoter regions. A further possibility for obtaining expression of an intrinsically disordered protein would be to introduce the host organism with a virus or bacteriophage containing a nucleic acid encoding a membrane protein.

Example 1
E. coli strain BL21 is prepared to contain a vector that produces an aquaporin protein linked to a His tag at the C-terminus. The His tag contains 10 contiguous histidine molecules attached to the primary sequence of aquaporin membrane proteins.

The Escherichia coli strain is cultured in a standard medium to obtain 150 L of total fermentation broth. The E. coli cells were recovered by filtering the fermentation broth with a microfiltration membrane having a pore size of 0.05 μm. After reducing the filtrate containing the E. coli cells to about 50 L, centrifuge at 5300 g for 20 minutes. Therefore, the E. coli cells are concentrated by microfiltration and the remaining medium is then removed as supernatant by centrifugation.

The pellet obtained by centrifugation is collected, and 1: 1 volume of 0.9% sodium chloride is added to wash the cells and dissolve the contaminating salt. Subsequently, the cleaning liquid is removed by a centrifuge running at 5300 g for 20 minutes. The supernatant was discarded and the washed cells were collected as pellets. The pellet can be cryopreserved at −20 ° C. or used directly in the next step.

Pellets containing E. coli cells were solubilized in approximately 47 L TRIS buffer used as binding buffer. After stirring for about 1 hour, 6.4 L of surfactant (5% LDAO) was added to solubilize the mixture to a final concentration of 0.6%. The mixture was incubated overnight at room temperature with gentle stirring. Cytolysis occurred by resuspending the cells in a buffer containing a detergent. Cytolysis releases the membrane protein from the inner membrane of the cell and solubilizes it with a detergent.

To remove negatively charged cell material, polyamines (Superfloc C581) were added in a volume of 427 mL. The mixture was incubated for 30 minutes with stirring at room temperature to coagulate negatively charged molecules such as cell debris, DNA, RNA, and some proteins. Membrane proteins solubilized by detergents remain in the aqueous phase. The mixture is centrifuged at a maximum speed of 5300 g for 15 minutes. Discard the pellet containing cell debris, DNA, RNA and solubilized protein and collect the supernatant. The supernatant is transferred to a container containing 107 L of dilution buffer to obtain a final volume of 160 L at a final concentration of 0.2% LDAO.

Prepare a column containing the GE Healthcare affinity resin "CaptoChelating". The resin is charged with ^{Ni 2+} that binds to the His10 tag. The column was loaded with diluted supernatant, followed by washing with a 10 column volume wash buffer containing 200 mM imidazole to wash away non-specific binding components. Subsequently, a 2.5 column volume elution buffer of aqueous 1000 mM imidazole was used to release the membrane protein from the column. Proteins eluted from the column were tested by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and showed predominantly a single band with a purity greater than 80%.

Example 2
E. coli strain BL21 is prepared to contain a vector that produces an aquaporin protein linked to a His tag at the C-terminus. The His tag contains 10 contiguous histidine molecules attached to the primary sequence of aquaporin membrane proteins.

The Escherichia coli strain is cultured in a standard medium to obtain 250 L of total fermentation broth. The E. coli cells were collected by filtering the fermentation broth through a PES flat sheet membrane with a pore size of 0.05 μm. The filtrate containing the E. coli cells is reduced to about 50 L and then centrifuged at 5300 g for 20 minutes using a Sorvall 16 L centrifuge. Therefore, the E. coli cells are concentrated by microfiltration and the remaining medium is then removed as supernatant by centrifugation. The pellet can be cryopreserved at −20 ° C. or used directly in the next step.

The pellet containing the E. coli cells was resuspended in about 50 L of buffer (aqueous solution of protease inhibitors PMSF and EDTA) and homogenized at 1000 bar using a Standed nm-GEN7575 homogenizer. Polyamines (Superfloc C581) are added at a concentration of 12 ml / L to separate the cell material of interest. The temperature was maintained at about 10-15 ° C. The mixture was incubated for 30 minutes with stirring at room temperature. The mixture was centrifuged at a maximum speed of 5300 g for 30 minutes. The pellet contains membrane protein and the supernatant is discarded.

The pellet was resuspended in 0.9% sodium chloride solution to a total protein concentration of about 50 mg / ml. Membrane protein solubilization was performed by adding 28 L of TRIS binding buffer and 4.5 liters of 5% LDAO to 5 L of resuspended pellet material. The mixture was incubated for 2-24 hours with gentle stirring at room temperature.

After the solubilization method, the mixture was centrifuged in a 2 L container at 5300 g for 90 minutes. The supernatant was collected and a dilution buffer was added to adjust the LDAO concentration to 0.2%.

Prepare a column containing the GE Healthcare affinity resin "CaptoChelating". The resin is charged with ^{Ni 2+} that binds to the His10 tag. The column was equilibrated by loading a binding buffer containing 0.2% LDAO. Subsequently, the diluted supernatant was loaded onto a column and washed with a 10 column volume wash buffer containing 200 mM imidazole to wash away non-specific binding components. Subsequently, a 2.5 column volume elution buffer of aqueous 1000 mM imidazole is used to release the membrane protein from the column. Proteins eluted from the column were tested by sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) chromatography and showed only a single band showing a purity greater than 95%.

Example 3
E. coli strain BL21 is prepared to contain a vector that produces an aquaporin protein linked to a His tag at the C-terminus. The His tag contains 10 contiguous histidine molecules attached to the primary sequence of aquaporin membrane proteins.

The E. coli strain is cultured in standard medium to obtain 250 L of total fermentation broth. The fermentation batch had OD600 13 at harvest and was induced for 42.5 hours. The material was homogenized twice at 100 MPa using a Stansted nm-GEN7575 homogenizer. 10 mL was removed from the dissolved material and added to a 15 mL Falcon tube.

The pellet was then separated from the supernatant by centrifugation at 5300 g for 1 hour. The pellet was then resuspended in 10 mL of 0.9% NaCl. The BCA assay was used to determine the concentration (mg / mL total protein) of the supernatant separated from the pellet resuspended after the first spin.

The supernatant separated from the resuspended pellet was finally solubilized in 0.6% LDAO for 2 hours (120 μL of Carbosynth's 5% LDAO stock was used in 1 mL of material) and centrifuged again at 5300 G for 15 minutes. Again, the supernatant was separated from the pellet. The pellet was resuspended in a LDAO-free binding buffer (again up to 1 mL).

Analysis of all samples with SDS-gel revealed that approximately 60% of the protein in the supernatant was aquaporin membrane protein.

Example 4
Following the purification of aquaporin Z in Example 2, the size distribution of solubilized proteins in LDAO was measured.

Comparisons were made between the same elution buffers with and without protein to assess whether the particle size distribution was affected by buffer components (including surfactants) or membrane proteins.

The aquaporin Z protein was eluted from the IMAC column using an elution buffer containing 1000 mM imidazole and 0.2% w / vLDAO. The purified protein was measured for its protein concentration by amino acid analysis. 6.44 mg / mL clear solubilized protein in elution buffer was loaded into three different cuvettes (Sarthtedt, 4 mL, PMMA, ca.no. 67.755, Nümbrecht, Germany) and (Malvern Instruments Ltd.). Malvern UK) Malvern Germany software v. The size distribution was analyzed with Malvern Zethasizer Nano-ZS using 7.02. The results shown in Table 1 are the average of the samples performed three times.

The elution buffer sample (without protein) contains a 97.9% population distribution of particles with a size of 8.6 nm or less, and the large detergent micelles retained by microfiltration are absent in solution. It is shown that. 81.4% of the particles in the sample containing the protein show a size of 108 nm or greater, indicating that the presence of the protein and detergent together form large soluble particles that are retained during microfiltration. ing. Surprisingly, this experiment shows that large, stable particles composed of LDAO micelles and membrane proteins are produced.

Example 5
Expression of histidine-tagged aquaporin from Oryza sativa Japonica (US) in Escherichia coli and its purification using IMAC The gene encoding the aquaporin of Oryza sativa Japonica (UNIPROT: A3C132) is used to improve expression in Escherichia coli. (A subsidiary of Thermo Fisher Scientific) Codon-optimized using the service. The obtained gene was synthesized by adding 10 histidine coding codons adjacent to the N-terminal and C-terminal, respectively, together with an NdeI / XhoI restriction site adjacent to the C-terminal (gene ID: aquaporin_Oryza_sativa_Japanica). Synthetic gene fragments were digested with NdeI / XhoI restriction enzymes and ligated into vector pUP1909 fragments digested and purified with NdeI / XhoI. The resulting ligation mixture was transformed into Escherichiacoli DH10B and kanamycin resistant transformants were selected on LB agar plates containing kanamycin. Transformants were confirmed by sequencing the gene construct. Subsequently, the isolated vector DNA was transferred to the production host Escherichia coli BL21.

Aquaporins in order to heterologous expression in E. coli, 30 g / L _{glycerol, 6g / L (NH 4)} 2 HPO 4, 3g / L KH 2 PO 4, 5g / L NaCl, 0.25g / L MgSO 4 · 7H 2 O The production host was grown in a minimal medium consisting of 0.4 g / L iron citrate (III) and 1 mL / L sterile filtered trace metal solution. The trace metals _{solution, 1g / L EDTA, 0.8g /} L CoCl 2 · 6H 2 O, 1.5 MnCl 2 · 4H 2 O, 0.4g / L CuCl 2 · 2H 2 O, 0.4g / L _{H 3 BO 3, 0.8g / L} Na 2 MoO 4 · 2H 2 O, made of _{1.3g / L Zn (CH 3 COO} ) 2 · 2H 2 O. After inoculation and overnight growth, further addition of MgSO ₄ · 7H ₂ O of 0.25 g / L.

E. coli was cultivated in 3L Applicon Bioreactors equipped with ez-Control by a batch fermentation method. Protein production was induced by adding IPTG at a final concentration of 0.5 mM and an optical density (OD of 600 nm) of about 30. Cultures were induced for about 24 hours and bacterial cells were harvested by centrifugation at 5300 g for 20 minutes.

Pellets containing E. coli cells were resuspended in a buffer (aqueous solution of protease inhibitors PMSF and EDTA) and homogenized at 1000 bar using a Standed nm-GEN7575 homogenizer. The temperature was maintained at about 10-15 ° C. The mixture was centrifuged at a maximum speed of 5300 g for 30 minutes. The pellet contains membrane protein and the supernatant is discarded.

The pellet was resuspended in 0.9% sodium chloride solution to a total protein concentration of about 50 mg / mL. Membrane protein solubilization was performed by adding 28 L of TRIS binding buffer and 4.5 liters of 5% LDAO to 5 L of resuspended pellet material. The mixture was incubated for 2-24 hours with gentle stirring at room temperature.

After the solubilization method, the mixture was centrifuged in a 2 L container at 5300 g for 90 minutes. The supernatant was collected and a dilution buffer was added to adjust the LDAO concentration to 0.2%.

After solubilization and clarification, the protein was captured using IMAC and eluted with an elution buffer containing 1000 mM imidazole and 0.2% w / v LDAO. Analysis of the elution fractions by SDSpage showed only a single major band that migrated at 27 kDa. This corresponds to the size of Japanese rice aquaporins. In addition, the results were confirmed by comparison with negative control purification from E. coli transformed with an empty vector. Negative control did not give purified protein. Western blot analysis with a histidine tag-specific antibody (TaKaRa Bio), as expected, gave a clear signal from the purified protein, from a negative control confirming the origin of the purified protein as a histidine-tagged membrane protein. There was no signal.

Example 6
Expression of histidine-tagged aquaporin from eucalyptus grandis in E. coli and its purification using IMAC The gene encoding eucalyptus grandis aquaporin (UNIPROT: A0A059C9Z4) is a Geneart (Thermo Fisher Scientific) subsidiary to improve expression in E. coli. Codon optimized using the service. The obtained gene was synthesized by adding 10 histidine coding codons adjacent to the N-terminal and C-terminal, respectively, together with an NdeI / XhoI restriction site adjacent to the C-terminal (gene ID: aquaporin_Eucalyptus_grandis). The gene was cloned and expressed as described in Example 5. The protein was successfully purified and confirmed as described in Example 5.

Example 7
Expression of histidine-tagged aquaporin from Solanum tuberosum (Potato in Denmark) in E. coli and its purification using IMAC The gene encoding the aquaporin of Solanum tuberosum (UNIPROT: Q38HT6) is used to improve expression in E. coli. Codon-optimized using the (Scientific subsidiary) service. The obtained gene was synthesized by adding 10 histidine coding codons adjacent to the N-terminal and C-terminal, respectively, together with an NdeI / XhoI restriction site adjacent to the C-terminal (gene ID: aquaporin_Solanum_tuberosum). The gene was cloned and expressed as described in Example 5. The protein was successfully purified and confirmed as described in Example 5.

Example 8
Expression of histidine-tagged aquaporin from Milnesium tardigradum (tardigrade) in E. coli and purification using IMAC The gene encoding aquaporin in Milnesium tardigradum (UNIPEROT: G5CTG2) is used to improve expression in Escherichia coli. Codon optimized using the (subsidiary) service. The obtained gene was synthesized by adding 10 histidine coding codons adjacent to the N-terminal and C-terminal, respectively, together with the NdeI / XhoI restriction site adjacent to the C-terminal (gene ID: aquaporin_Mirnesium_tardigradum). The gene was cloned and expressed as described in Example 5. The protein was successfully purified and confirmed as described in Example 5.

Example 9
Expression of histidine-tagged aquaporin from Halomonas in E. coli and its purification using IMAC The gene encoding Halomonas aquaporin (UNIPROT: A0A2N0G6U6) is a Geneart (Thermo Fisher Scientific subsidiary) to improve expression in E. coli. ) Codon optimized using the service. The obtained gene was synthesized by adding 10 histidine coding codons adjacent to the N-terminal and C-terminal, respectively, together with an NdeI / XhoI restriction site adjacent to the C-terminal (gene ID: aquaporin_Halomonas_sp). The gene was cloned and expressed as described in Example 5. The protein was successfully purified and confirmed as described in Example 5.

Example 10
Expression of histidine-tagged aquaporin from Kyrpidiaspormannii in E. coli and its purification using IMAC The gene encoding aquaporin from the genus Kyrpidia (UNIPROT: A0A2K8N5Z5) is used to improve expression in Escherichia coli. Codon optimized using the service. The obtained gene was synthesized by adding 10 histidine coding codons adjacent to the N-terminal and C-terminal, respectively, together with an NdeI / XhoI restriction site adjacent to the C-terminal (gene ID: aquaporin_Kyrpidia_spormannii). The gene was cloned and expressed as described in Example 5. The protein was successfully purified and confirmed as described in Example 5.

Example 11
Expression of histidine-tagged aquaporin from the genus Thermothermovator in Escherichia coli and its purification using IMAC The gene encoding aquaporin from the genus Thermothermovator (UNIPROT: A0A223ZCQ2) is used to improve the expression of HistidineA223ZCQ2 in Escherichia coli. Codon-optimized using the (subsidiary) service. The obtained gene was synthesized by adding 10 histidine coding codons adjacent to the N-terminal and C-terminal, respectively, together with an NdeI / XhoI restriction site adjacent to the C-terminal (gene ID: aquaporin_Methanothermobacter_sp). The gene was cloned and expressed as described in Example 5. The protein was successfully purified and confirmed as described in Example 5.

Example 12
Expression of histidine-tagged aquaporin from Novicabilus thermophilus in E. coli and its purification using IMAC The gene encoding aquaporin in Novivacillus thermofilus (UNIPROT: A0A1U9K5R2) uses the Geneart Codon-optimized to improve expression. The obtained gene was synthesized by adding 10 histidine coding codons adjacent to the N-terminal and the C-terminal, respectively, together with the NdeI / XhoI restriction site adjacent to the C-terminal (gene ID: aquaporin_Novicabilus_thermofilus). The gene was cloned and expressed as described in Example 5. The protein was successfully purified and confirmed as described in Example 5.

Example 13
Expression of outer membrane protein A (OmpA) from Escherichia coli in E. coli Expression and purification of OmpA (UNIPROT: P0A910) in E. coli except that E. coli was transformed with an empty vector and solubilization was extended to 24 hours. In essence, it was carried out as described in Example 5. The resulting solubilized protein was estimated to be about 30-50% pure by SDS-PAGE, thus clearly demonstrating successful isolation of OpPA by membrane fraction according to standard purification procedures. .. An additional size exclusion chromatography (SEC) step can further purify OpPA.

Example 14
A Saccharomyces cerevisiae strain expressing aquaporin-Z (AqpZ) is constructed from Escherichia coli isolated by a tobacco etch virus (TEV) protease cleavage site by fusing to N-terminal histidine-tagged yEGFP.

AqpZ (UNIPROT ID: P60844) derived from Escherichia coli is described in S. coli. Using Geneart's services to improve expression in S. cerevisiae, S. cerevisiae. Codon-optimized for expression in S. cerevisiae. S. For expression in S. cerevisiae, AqpZ was fused with yeast-enhanced green fluorescent protein (yEGFP) at the N-terminus (Brendan P. Cormac et al., Microbiology (1997), 143, 303-311) with visual detection of membrane proteins. It enabled quantification. In addition, eight histidines (His8) were added to the N-terminus of yEGFP as an IMAC purification tag. His8-yEGFP and AqpZ were genetically isolated by a TEV protease cleavage site incorporated by PCR primers during construction.

Rapid and efficient construction of plasmids encoding His8-yEGFP-TEV-AqpZ fusion is described in (Scientific Reports 7:16899), His8-yEGFP-TEV PCR fragment, TEV-AqpZ PCR fragment, And S. cerevisiae between SalI, HindIII, and BamHI digestion-derived linearized expression plasmids of the pEMBLyex4 plasmid. It was performed by in vivo homologous recombination of overlapping regions incorporated with S. cerevisiae primers. The TEV cleavage site allows subsequent removal of the His8-yEGFP protein by TEV protease.

S. Selection of S. cerevisiae transformants was performed on minimal medium plates supplemented with leucine and lysine to ensure survival of bacterial cells with uracil-deficient but correctly recombined DNA fragments. The medium composition and amino acid concentration used in this example were the same as those listed in Example 15.

Example 15
Expression of His8-yEGFP-TEV-AqpZ with Saccharomyces cerevisiae Selective single colonies of transformed yeast cells until saturated with 5 mL glucose minimal medium supplemented with 60 mg / L leucine and 30 mg / L lysine. Was propagated to. Subsequently, 200 μL of this culture was grown in 5 mL glucose minimal medium supplemented with 30 mg / L lysine for selection of high plasmid copy numbers. A frozen stock of high plasmid copy number cells was prepared.

200 μL of thawed frozen stock was added to 10 mL of minimal medium supplemented with lysine and grown to saturation. 1 mL of culture was transferred to 100 mL of the same medium. After overnight growth, an aliquot corresponding to 0.05 final OD600 was added to a minimum medium of 1.5 liters containing an initial concentration of 20 g / L glucose as a carbon source and 30 g / L glycerol supplemented with additional amino acids. Moved. Cultures were cultured in a 3LAplikon® bioreactor equipped with an ez-Control connected to a PC running Lucullus® software (Applikon, The Netherlands, and SecureCell, Switzerland).

The first part of fermentation was carried out in minimal medium at 20 ° C. The bioreactor was supplied with glucose to a final concentration of 3% w / v when the initial amount of glucose was metabolized. The pH of the growth medium was maintained at 6.0 with a computer-controlled addition of _{1 M NH 4 OH. When CO 2} emissions leveled off due to restricted access to glucose, the bioreactor was cooled to 15 ° C. before inducing recombinant AQP production. Expression of the recombinant protein was initiated by the addition of a 50 mL / L expression medium consisting of 400 mL / L ASD-10, 400 mL / L excess amino acid, 200 g / L glycerol, and 20 g / L galactose. Yeast cells were collected after about 96 hours.

The minimum medium consisted of 20 g / L glucose, 100 mL / L ASD-10, 5 mL / L V-200, 30 g / L glycerol, and 0.1 g / L CaCl ₂ . The ASD-10 _{is, 50g / L (NH 4)} 2 SO 4, 8.75g / L KH 2 PO 4, 1.25g / L K 2 HPO 4, 5g / L MgSO 4 · 7H 2 O, 1g / L _{NaCl, 5mg / L H 3 BO} 3, 1mg / L KI, 4mg / L MnSO 4 · 1H 2 O, 4.2mg / L ZnSO 4 · 7H 2 O, 0.4mg / L CuSO 4 · 5H 2 O, 2mg / L FeCl _3, composed of _{2mg / L Na 2 MoO 4 ·} 2H 2 O. The V-200 is 4 mg / L biotin, 400 mg / L D-pantothenic acid, 0.4 mg / L folic acid, 2000 mg / L myo-inositol, 80 mg / L niacin, 40 mg / L p-aminobenzoic acid, 80 mg / L pyridoxine. , 40 mg / L riboflavin, 80 mg / L thiamine. As described, the medium contains 600 mg / L alanine, 600 mg / L arginine, 600 mg / L cysteine, 3000 mg / L glutamic acid, 2000 mg / L lysine, 600 mg / L methionine, 1500 mg / L phenylalanine, 600 mg / L proline. It contained additional amino acids consisting of 10000 mg / L serine, 900 mg / L tyrosine, 4500 mg / L valine, 2000 mg / L aspartic acid, 4000 mg / L threonine, 600 mg / L histidine, and 600 mg / L tryptophan.

Example 16
Cloning of His8-yEGFP-TEV-AQP5 from Milnesium tardigradum in a bioreactor containing Saccharomyces cerevisiae The His8-yEGFP-TEV-AQP5 construct was prepared according to the procedure outlined in Example 14. M. The tardigradum-derived AQP5 protein (UNIPROT: G5CTG2) was a codon optimized for expression in E. coli, despite the need for expression in yeast.

Example 17
S. Purification of His8-yEGFP-TEV-AqpZ and His8-yEGFP-TEV-AQP5 from S. cerevisiae S. cerevisiae. Purification of heterologously expressed membrane proteins from S. cerevisiae is described in Example 2, except that the amount of buffer applied was reduced to match the reduced culture volume and lysis was performed at 1800 bar. I ran as it was. Protein production and purity were successfully confirmed on both SDS page and Western blot fusion proteins, with no contaminating proteins detected.

Claims (26)

The following steps:
a. Expressing membrane proteins in a host organism present in an aqueous medium,
b. To release the membrane protein from the host organism,
c. To solubilize the membrane protein by adding a detergent solution,
d. Collecting the liquid fraction of the solubilized membrane protein as a supernatant by centrifugation,
e. The liquid fraction is subjected to chromatography to bind or retain the membrane protein on a stationary phase, and
f. To produce the membrane protein by eluting the stationary phase with an elution buffer.
A method for producing a membrane protein, which comprises the above method and centrifuges in step d at 500 g to 30,000 g. The production method according to claim 1, wherein the aqueous medium containing the host organism in step a is filtered before the host organism is released by step b. The production method according to claim 2, wherein the aqueous medium containing the host organism in step a is filtered through a microfiltration membrane having a pore size of 0.5 μm or less. The production method according to any one of claims 1 to 3, wherein the host organism is optionally filtered and then isolated by centrifugation of the aqueous medium containing the host organism. The production method according to any one of claims 1 to 4, wherein the centrifugation is carried out at 500 g to 30,000 g, preferably 1,000 g to 10,000 g. The isolated host organism is washed with isotonic saline to dissolve the contaminating salt, and then centrifuged to isolate the washed host organism according to any one of claims 1 to 5. The manufacturing method described. The production method according to any one of claims 1 to 6, wherein a dilution buffer is added before step b. The production method according to any one of claims 1 to 7, wherein an aqueous solution is added to release the membrane protein from the host organism. The production method according to any one of claims 1 to 8, wherein the aqueous lysis buffer is a surfactant solution, and the surfactant solution simultaneously solubilizes the membrane protein. The production method according to any one of claims 1 to 9, wherein the host cell can be affected by the surfactant during stirring. The production method according to any one of claims 1 to 7, wherein the release of the membrane protein from the host cell is carried out using a homogenizer. The production method according to any one of claims 1 to 11, wherein a cationic flocculant is added to the liberated membrane protein to form a suspension. The production method according to any one of claims 1 to 12, wherein the cationic flocculant is a polyamine compound such as Superfloc C581. The production method according to any one of claims 1 to 13, wherein the cationic flocculant can react with the cell portion of the resuspension to form flocs while being gently stirred. The production method according to any one of claims 1 to 14, wherein the liquid fraction of step d is recovered as a supernatant from centrifugation of a suspension containing flocs. The liquid fraction of step d is recovered from the resuspension of the solid fraction, and the solid fraction is resuspended in a surfactant solution for solubilizing the membrane protein, according to any one of claims 1 to 7. The manufacturing method described. The production method according to claim 16, wherein the liquid fraction of step d is recovered as a supernatant by centrifugation. Any one of claims 1 to 17, wherein the surfactant is selected from the group consisting of lauryldimethylamine N-oxide (LDAO), octyl glucoside (OG), dodecyl maltoside (DDM) or a combination thereof. The manufacturing method described in. The production method according to any one of claims 1 to 18, wherein the centrifugation is carried out at 500 g to 30,000 g, preferably 1,000 g to 10,000 g. The production method according to any one of claims 1 to 19, wherein a stationary phase capable of binding or retaining the membrane protein is present in the column. The production method according to any one of claims 1 to 20, wherein the membrane protein associates with the first portion of the affinity pair and the stationary phase associates with the second portion of the affinity pair. The production method according to any one of claims 1 to 21, wherein the first portion of the affinity pair is a histidine tag. The production method according to any one of claims 1 to 22, wherein the histidine tag contains eight or more histidine molecules. The production method according to any one of claims 1 to 23, wherein the elution buffer contains imidazole. The production according to any one of claims 1 to 24, wherein the column before elution with the elution buffer is washed with a washing buffer containing imidazole having a concentration of 40% or less in the elution buffer. Method. The production method according to any one of claims 1 to 25, wherein the concentration of imidazole in the elution buffer is 400 mM or more.